N-maleimidyl polymer derivatives

ABSTRACT

The invention is directed to multi-functional N-maleimidyl polymer derivatives comprising a water soluble and non-peptidic polymer backbone having a terminal carbon, such as a poly(alkylene glycol), the terminal carbon of the polymer backbone being directly bonded to the nitrogen atom of a N-maleimidyl moiety without a linking group therebetween. The invention also provides two methods of preparing such linkerless N-maleimidyl polymer derivatives.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/294,148, filed Oct. 14, 2016, now U.S. Pat. No. 9,828,465, which is acontinuation of U.S. patent application Ser. No. 14/865,362, filed Sep.25, 2015, now U.S. Pat. No. 9,493,609, which is a continuation of U.S.patent application Ser. No. 14/627,917, filed Feb. 20, 2015, now U.S.Pat. No. 9,169,350, which is a continuation of U.S. patent applicationSer. No. 14/283,788, filed May 21, 2014, now U.S. Pat. No. 8,992,902,which is a continuation of U.S. patent application Ser. No. 13/890,799,filed May 9, 2013, now U.S. Pat. No. 8,765,111, which is a continuationof U.S. patent application Ser. No. 11/134,076, filed May 20, 2005, nowU.S. Pat. No. 8,454,946, which is a continuation of U.S. patentapplication Ser. No. 10/408,834, filed Apr. 7, 2003, now U.S. Pat. No.6,911,197, which is a continuation of U.S. patent application Ser. No.09/790,250, filed Feb. 21, 2001, now U.S. Pat. No. 6,602,498, whichclaims the benefit of priority to U.S. Provisional Patent ApplicationNo. 60/183,833, filed on Feb. 22, 2000, all of which are incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to N-maleimidyl derivatives of water-soluble andnon-peptidic polymers.

BACKGROUND OF THE INVENTION

Covalent attachment of the hydrophilic polymer poly(ethylene glycol),abbreviated PEG, also known as poly(ethylene oxide), abbreviated PEO, tomolecules and surfaces is of considerable utility in biotechnology andmedicine. In its most common form, PEG is a linear polymer terminated ateach end with hydroxyl groups:HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH

The above polymer, alpha-,omega-dihydroxylpoly(ethylene glycol), can berepresented in brief form as HO-PEG-OH where it is understood that the-PEG- symbol represents the following structural unit:—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—where n typically ranges from about 3 to about 4000.

PEG is commonly used as methoxy-PEG-OH, or mPEG in brief, in which oneterminus is the relatively inert methoxy group, while the other terminusis a hydroxyl group that is subject to ready chemical modification. Thestructure of mPEG is given below.CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OH

Random or block copolymers of ethylene oxide and propylene oxide, shownbelow, are closely related to PEG in their chemistry, and they can besubstituted for PEG in many of its applications.HO—CH₂CHRO(CH₂CHRO)_(n)CH₂CHR—OHwherein each R is independently H or CH₃.

PEG is a polymer having the properties of solubility in water and inmany organic solvents, lack of toxicity, and lack of immunogenicity. Oneuse of PEG is to covalently attach the polymer to insoluble molecules tomake the resulting PEG-molecule “conjugate” soluble. For example, it hasbeen shown that the water-insoluble drug paclitaxel, when coupled toPEG, becomes water-soluble. Greenwald, et al, J. Org Chem., 60:331-336(1995).

To couple PEG to a molecule, such as a protein, it is often necessary to“activate” the PEG by preparing a derivative of the PEG having afunctional group of terminus thereof. The functional group is chosenbased on the type of available reactive group on the molecule that willbe coupled to the PEG. For example, the functional group could be chosento react with an amino group on a protein in order to form a PEG-proteinconjugate. There is a continuing need in the art for new activated PEGderivatives useful for coupling to biologically active molecules.

SUMMARY OF THE INVENTION

The invention provides multi-functional N-maleimidyl polymerderivatives, such as bifunctional and multi-arm N-maleimidyl PEGderivatives, and methods for preparing such derivatives. The derivativesof the invention have no linking group between the terminus of thepolymer backbone and the nitrogen atom of the maleimidyl moiety. Theabsence of a linker minimizes structural complexity of the derivativeand simplifies synthesis of the derivative. Further, the “linkerless”derivatives of the invention typically cost less to produce and exhibitreduced likelihood of degradation in vivo. Such maleimidyl-activatedpolymers are suitable for coupling to other molecules bearing thiolgroups, including, but not limited to, proteins having one or morecysteine thiol groups.

The invention provides a multi-functional N-maleimidyl polymerderivative comprising a water soluble and non-peptidic polymer backbonehaving an average molecular weight from about 800 Da to about 100,000Da, the polymer backbone having a first terminus bonded to a firstfunctional group and a second terminus having a terminal carbon, whereinsaid terminal carbon of said second terminus is directly bonded to aN-maleimidyl moiety having the structure:

wherein L is the point of bonding to the terminal carbon of the secondterminus of the polymer backbone. The second functional group may be asecond maleimidyl moiety or any other functional group known in the artthat will not react with a maleimidyl group. The polymer backbone hastwo or more termini “activated” with a functional group such as amaleimidyl group.

The polymer backbone is preferably a poly(alkylene glycol), copolymerthereof, terpolymer thereof, or mixture thereof. Examples includepoly(ethylene glycol), poly(propylene glycol), and copolymers ofethylene glycol and propylene glycol. As explained in greater detailbelow, preferred embodiments of the invention utilize PEG polymers, suchas bifunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendentPEG, and PEG with degradable linkages therein.

The invention provides two methods of preparing the linkerlessN-maleimidyl polymer derivatives. In one method, a water-soluble andnon-peptidic polymer backbone having an average molecular weight fromabout 800 Da to about 100,000 Da, the polymer backbone having a firstterminus bonded to a first functional group and a second terminus bondedto an amine group, is reacted with maleic anhydride to form an open ringintermediate. The open ring intermediate is heated in the presence ofacetic anhydride and a salt of acetic acid, such as sodium or potassiumacetate, to form a multi-functional N-maleimidyl polymer derivativeproduct. In a second method, an N-alkoxycarbonylmaleimide is reactedwith the water-soluble and non-peptidic polymer backbone having anaverage molecular weight of about 800 Da to about 100,000 Da and aterminal amine group to form the N-maleimidyl polymer derivative productin a single step. In one embodiment, the polymer backbone used in eitherreaction method is X-PEG-NH₂, wherein PEG is poly(ethylene glycol) and Xis a second functional group.

Using either method, the N-maleimidyl polymer derivative product can bepurified prior to use. For example, ion exchange chromatography andprecipitation techniques can be employed to purify the final product.The N-maleimidyl polymer derivative product can be reacted with abiologically active agent to form a biologically active polymerconjugate. As noted above, the N-maleimidyl polymer derivatives areparticularly suited for reaction with thiol groups, such as thiol groupson proteins or peptides.

DETAILED DESCRIPTION OF THE INVENTION

The terms “functional group”, “active moiety”, “activating group”,“reactive site”, “chemically reactive group” and “chemically reactivemoiety” are used in the art and herein to refer to distinct, definableportions or units of a molecule. The terms are somewhat synonymous inthe chemical arts and are used herein to indicate the portions ofmolecules that perform some function or activity and are reactive withother molecules. The term “active”, when used in conjunction withfunctional groups, is intended to include those functional groups thatreact readily with electrophilic or nucleophilic groups on othermolecules, in contrast to those groups that require strong catalysts orhighly impractical reaction conditions in order to react. For example,as would be understood in the art, the term “active ester” would includethose esters that react readily with nucleophilic groups such as amines.Typically, an active ester will react with an amine in aqueous medium ina matter of minutes, whereas certain esters, such as methyl or ethylesters, require a strong catalyst in order to react with a nucleophilicgroup. Due to its relatively inert nature, an alkoxy group is notconsidered a functional group herein.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pHs, e.g., under physiological conditions for anextended period of time, perhaps even indefinitely. Hydrolyticallyunstable or degradable linkages means that the linkages are degradablein water or in aqueous solutions, including for example, blood.Enzymatically unstable or degradable linkages means that the linkage canbe degraded by one or more enzymes. As understood in the art, PEG andrelated polymers may include degradable linkages in the polymer backboneor in the linker groups between the polymer backbone and one or more ofthe terminal functional groups of the polymer molecule. For example,ester linkages formed by the reaction of PEG carboxylic acids oractivated PEG carboxylic acids with alcohol groups on a biologicallyactive agent generally hydrolyze under physiological conditions torelease the agent. Other hydrolytically degradable linkages includecarbonate linkages; imine linkages resulted from reaction of an amineand an aldehyde (see, e.g., Ouchi et al., Polymer Preprints, 38(1):582-3(1997), which is incorporated herein by reference); phosphate esterlinkages formed by reacting an alcohol with a phosphate group; hydrazonelinkages which are reaction product of a hydrazide and an aldehyde;acetal linkages that are the reaction product of an aldehyde andalcohol; orthoester linkages that are the reaction product of a formateand an alcohol; peptide linkages formed by an amine group, e.g., at anend of a polymer such as PEG, and a carboxyl group of a peptide; andoligonucleotide linkages formed by a phosphoramidite group, e.g., at theend of a polymer, and a 5′ hydroxyl group of an oligonucleotide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalorganism, including but not limited to viruses, bacteria, fungi, plants,animals, and humans. In particular, as used herein, biologically activemolecules include any substance intended for diagnosis, cure mitigation,treatment, or prevention of disease in humans or other animals, or tootherwise enhance physical or mental well-being of humans or animals.Examples of biologically active molecules include, but are not limitedto, peptides, proteins, enzymes, small molecule drugs, dyes, lipids,nucleosides, oligonucleotides, cells, viruses, liposomes, microparticlesand micelles. Classes of biologically active agents that are suitablefor use with the invention include, but are not limited to, antibiotics,fungicides, anti-viral agents, anti-inflammatory agents, anti-tumoragents, cardiovascular agents, anti-anxiety agents, hormones, growthfactors, steroidal agents, and the like.

The terms “alky,” “alkene,” and “alkoxy” include straight chain andbranched alkyl, alkene, and alkoxy, respectively. The term “lower alkyl”refers to C1-C6 alkyl. The term “alkoxy” refers to oxygen substitutedalkyl, for example, of the formulas —OR or —ROR¹, wherein R and R¹ areeach independently selected alkyl. The terms “substituted alkyl” and“substituted alkene” refer to alkyl and alkene, respectively,substituted with one or more non-interfering substituents, such as butnot limited to, C3-C6 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and thelike; acetylene; cyano; alkoxy, e.g., methoxy, ethoxy, and the like;lower alkanoyloxy, e.g., acetoxy; hydroxy; carboxyl; amino; loweralkylamino, e.g., methylamino; ketone; halo, e.g., chloro or bromo;phenyl; substituted phenyl, and the like. The term “halogen” includesfluorine, chlorine, iodine and bromine.

“Aryl” means one or more aromatic rings, each of 5 or 6 carbon atoms.Multiple aryl rings may be fused, as in naphthyl or unfused, as inbiphenyl. Aryl rings may also be fused or unfused with one or morecyclic hydrocarbon, heteroaryl, or heterocyclic rings.

“Substituted aryl” is aryl having one or more non-interfering groups assubstituents.

“Non-interfering substituents” are those groups that yield stablecompounds. Suitable non-interfering substituents or radicals include,but are not limited to. halo, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, alkoxy, C₇-C₁₂ aralkyl, C₇-C₁₂ alkaryl, C₃-C₁₀ cycloalkyl,C₃-C₁₀ cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylenyl,biphenyl, C₂-C₁₂ alkoxyalkyl, C₇-C₁₂ alkoxyaryl, C₇-C₁₂ aryloxyalkyl,C₆-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀ alkylsulfonyl,—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8, aryl, substitutedaryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substitutedheterocyclic radical, nitroalkyl, —NO₂, —CN, —NRC(O)—(C₁-C₁₀ alkyl),—C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ thioalkyl, —C(O)O—(C₁-C₁₀ alkyl), —OH,—SO₂, ═S, —COOH, —NR₂, carbonyl, —C(O)—(C₁-C₁₀ alkyl)-CF₃, —C(O)—CF₃,—C(O)NR₂, alkyl)-S—(C₆-C₁₂ aryl), —C(O)—(C₆-C₁₂ aryl),—(CH₂)_(m)—O—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein each m is from 1 to 8,—C(O)NR₂, —C(S)NR₂, —SO₂NR₂, —NRC(O)NR₂, —NRC(S)NR₂, salts thereof, andthe like. Each R as used herein is H, alkyl or substituted alkyl, arylor substituted aryl, aralkyl, or alkaryl.

The invention provides a multi-functional N-maleimidyl polymerderivative comprising a water soluble and non-peptidic polymer backbonehaving an average molecular weight from about 800 Da to about 100,000Da, the polymer backbone having a first terminus bonded to a firstfunctional group and a second terminus having a terminal carbon, whereinsaid terminal carbon of said second terminus is directly bonded to aN-maleimidyl moiety having the structure:

wherein L is the point of bonding to the terminal carbon of the secondterminus of the polymer backbone.

The polymer backbone of the water-soluble and non-peptidic polymer canbe poly(ethylene glycol) (i.e. PEG). However, it should be understoodthat other related polymers are also suitable for use in the practice ofthis invention and that the use of the term PEG or poly(ethylene glycol)is intended to be inclusive and not exclusive in this respect. The termPEG includes poly(ethylene glycol) in any of its forms, includingbifunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG(i.e. PEG or related polymers having one or more functional groupspendent to the polymer backbone), or PEG with degradable linkagestherein.

PEG is typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects. PEGhaving the formula —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about3 to about 4000, typically from about 20 to about 2000, is one usefulpolymer in the practice of the invention. PEG having a molecular weightof from about 800 Da to about 100,000 Da are particularly useful as thepolymer backbone.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol oligomers, pentaerythritol andsorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(-PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. No. 5,932,462, which is incorporated byreference herein in its entirety, can also be used as the polymerbackbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(-YCHZ2)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

It is understood by those skilled in the art that the term poly(ethyleneglycol) or PEG represents or includes all the above forms.

Many other polymers are also suitable for the invention. Polymerbackbones that are non-peptidic and water-soluble, with from 2 to about300 termini, are particularly useful in the invention. Examples ofsuitable polymers include, but are not limited to, other poly(alkyleneglycols), such as poly(propylene glycol) (“PPG”), copolymers thereof(e.g. copolymers of ethylene glycol and propylene glycol), terpolymersthereof, mixtures thereof, and the like. Although the molecular weightof each chain of the polymer backbone can vary, it is typically in therange of from about 800 Da to about 100,000 Da, often from about 6,000Da to about 80,000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble and non-peptidic polymer backbonesis by no means exhaustive and is merely illustrative, and that allpolymeric materials having the qualities described above arecontemplated.

The polymer derivatives of the invention are “multi-functional”, meaningthat the polymer backbone has at least two termini, and possibly as manyas about 300 termini, functionalized or activated with a functionalgroup. Multifunctional polymer derivatives include linear polymershaving two termini, each terminus being bonded to a functional groupwhich may be the same or different.

In one embodiment, the polymer derivative has the structure:

wherein:

POLY is a linear water soluble and non-peptidic polymer having aterminal carbon (e.g., PEG), the terminal carbon being directly bondedto the nitrogen atom of the N-maleimidyl moiety; and

X is a second functional group.

Examples of suitable functional groups for use as X include hydroxyl,protected hydroxyl, active ester, such as N-hydroxysuccinimidyl estersand 1-benzotriazolyl esters, active carbonate, such asN-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates,acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, protected amine, hydrazide, protectedhydrazide, protected thiol, carboxylic acid, protected carboxylic acid,isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine,vinylpyridine, iodoacetamide, epoxide, glyoxals, diones, mesylates,tosylates, and tresylate. The functional group is typically chosen forattachment to a functional group on a biologically active agent. Aswould be understood, the selected X moiety should be compatible with themaleimidyl group so that reaction with the maleimidyl group does notoccur. Particularly preferred functional groups include —OH, —NH₂,—CO₂H, —CHO, —CH(OC₂H₅)₂, N-hydroxysuccinimidyl esters, 1-benzotriazolylesters, N-hydroxysuccinimidyl carbonates, 1-benzotriazolyl carbonates,and tresylate. The N-maleimidyl polymer derivatives may behomobifunctional, meaning that the second functional group (i.e., X) isalso N-maleimidyl moiety, or heterobifunctional, meaning that the secondfunctional group is a different functional group.

As would be understood in the art, the term “protected” refers to thepresence of a protecting group or moiety that prevents reaction of thechemically reactive functional group under certain reaction conditions.The protecting group will vary depending on the type of chemicallyreactive group being protected. For example, if the chemically reactivegroup is an amine or a hydrazide, the protecting group can be selectedfrom the group of tert-butyloxycarbonyl (t-Boc) and9-fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is athiol, the protecting group can be orthopyridyldisulfide. If thechemically reactive group is a carboxylic acid, such as butanoic orpropionic acid, or a hydroxyl group, the protecting group can be benzylor an alkyl group such as methyl, ethyl, or tert-butyl. Other protectinggroups known in the art may also be used in the invention.

Specific examples of terminal functional groups in the literatureinclude N-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698,5,468,478), amine (see e.g., Buckmann et al. Makromol. Chem. 182:1379(1981), Zalipsky et al. Eur. Polym J. 19:1177 (1983)), hydrazide (Seee.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidylpropionate and succinimidyl butanoate (see, e.g., Olson et al. inPoly(ethylene glycol) Chemistry & Biological Applications, pp 170-181,Harris & Zalipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat.No. 5,672,662), succinimidyl succinate (See, e.g., Abuchowski et al.Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al. Macrolol. Chem.180:1381 (1979), succinimidyl ester (see, e.g., U.S. Pat. No.4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No.5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J. Biochem.94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354 (1991),oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal. Biochem.131:25 (1983), Tondelli et al., J. Controlled Release 1:251 (1985)),p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl. Biochem.Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech.,27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem.Ed. 22:341 (1984), U.S. Pat. Nos. 5,824,784, 5,252,714), maleimide (see,e.g., Goodson et al. Bio/Technology 8:343 (1990), Romani et al. inChemistry of Peptides and Proteins 2:29 (1984)), and Kogan, SyntheticComm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g., Woghiren, etal. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g., Sawhney et al.,Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g., U.S. Pat. No.5,900,461). All of the above references are incorporated herein byreference.

In a preferred embodiment, the polymer derivatives of the inventioncomprise a polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂-Lwherein:

L is the point of bonding to the nitrogen atom of the N-maleimidylmoiety;

X is a functional group as described above; and

n is about 20 to about 4000.

A specific example of a homobifunctional polymer derivative comprisingthe above polymer backbone is shown below:

wherein n is about 20 to about 4000.

One example of a multi-armed embodiment of the invention has thefollowing structure:

wherein:

POLY is a water-soluble and non-peptidic polymer backbone having aterminal carbon (e.g., PEG), the terminal carbon being directly bondedto the nitrogen atom of the N-maleimidyl moiety;

R is a central core molecule, such as glycerol, glycerol oligomers,pentaerythritol, sorbitol, or lysine; and

q is an integer from 2 to about 300.

The derivatives of the invention can be prepared by two methods. In thefirst method, a water-soluble and non-peptidic polymer backbone havingan average molecular weight from about 800 Da to about 100,000 Da, thepolymer backbone having a first terminus bonded to a first functionalgroup and a second terminus bonded to an amine group, is reacted withmaleic anhydride to form an open ring amide carboxylic acidintermediate. The ring of the intermediate is then closed in a secondstep by heating the intermediate in the presence of acetic anhydride anda salt acetic acid, such as sodium or potassium acetate. Preferably, theheating step comprises heating the intermediate at a temperature ofabout 50° C. to about 140° C. for about 0.2 to about 5 hours. This twostep process is summarized in the reaction scheme below:

Step 1

Step 2

As shown, a preferred polymer backbone for use in the reaction has theformula X-PEG-NH₂, wherein PEG is poly(ethylene glycol) and X is afunctional group which does not react with amine or maleimidyl groups.Examples of suitable functional groups include hydroxyl, protectedhydroxyl, acetal, alkenyl, amine, protected amine, protected hydrazide,protected thiol, carboxylic acid, protected carboxylic acid, maleimide,dithiopyridine, and vinylpyridine. The open ring intermediate ispreferably purified by precipitation prior to the heating step.

The crude N-maleimidyl polymer derivative product of the above reactionscheme may contain a substantial amount of the open ring intermediate.As a result, it is generally preferable to purify the polymer derivativeproduct. Preferred purification techniques include precipitation and ionexchange chromatography.

In one embodiment, the purification step comprises passing theN-maleimidyl polymer derivative product through an ion exchange column,collecting an eluent containing the N-maleimidyl polymer derivativeproduct from the column, and precipitating the N-maleimidyl polymerderivative product by contacting the product with a solvent, such asethyl ether, isopropanol, or mixtures thereof. The precipitated productmay then be collected by filtration and dried.

In a second preferred method for preparation of the N-maleimidyl polymerderivatives of the invention in a single step, anN-alkoxycarbonylmaleimide is reacted with a water-soluble andnon-peptidic polymer backbone having an average molecular weight fromabout 800 Da to about 100,000 Da and a terminal amine group to form aN-maleimidyl polymer derivative product. An exemplary reaction scheme isshown below:

wherein PEG is poly(ethylene glycol) and X is a capping group such asalkoxy or a functional group as described above.

Purification of the crude product can also be accomplished byprecipitation and ion exchange chromatography as described above.

Heterobifunctional derivatives of this invention can be prepared by thereactions described above by utilizing appropriately substitutedheterobifunctional polymer amines. An example of a heterobifunctionalpolymer amine is a PEG amine acid:NH₂-PEG-CO₂HAnother example is PEG diamine, in which one of the amines is protectedby a moiety such as t-Boc:NH₂-PEG-NH-t-Boc

After conversion of the amine group to the maleimidyl group, theresulting N-maleimidyl PEG heterobifunctional molecule can then beconverted to other useful heterobifunctional N-maleimidyl PEGderivatives. For example, a α-N-maleimidyl, co-carboxylic acid PEG canbe converted to the N-succinimidyl ester. In another example, t-Boc canbe hydrolyzed to yield an ω-amino-α-N-maleimidyl PEG.

Heterobifunctional derivatives are useful when it is desired to attachdifferent molecules to each terminus of the polymer. For example, theα-N-maleimidyl-ω-N-succinimidyl carboxylate PEG would allow theattachment of a molecule having a thiol group to the N-maleimidylterminus of the PEG and a molecule having an amino group to theN-succinimidyl carboxylate terminus of the PEG.

The N-maleimidyl polymer derivatives of the invention can be used toreact with a biologically active agent, such as a protein or peptide, toform a biologically active polymer conjugate. Since the resultingconjugate does not contain a linker between the maleimidyl moiety andthe polymer terminus, there is less likelihood of degradation of theconjugate in vivo, thereby providing a more hydrolytically stablebiologically active polymer conjugate.

The following examples are given to illustrate the invention, but shouldnot be considered in limitation of the invention.

Example 1 Preparation of N-Methoxy PEG₅₀₀₀ Maleimide

mPEG-NH₂ (ave. MW=5000 Da, 1 g) was dissolved in saturated aqueousNaHCO₃ (5 ml) and the mixture was cooled to 0° C.N-methoxycarbonylmaleimide (0.1 g) was added with vigorous stirring.After stirring for 10 minutes, water (10 ml) was added and the mixturewas stirred an additional 45 minutes. The pH was adjusted to 3.0 with0.5 N sulfuric acid and about 15 wt % NaCl was added. The reaction wasextracted with CH₂Cl₂ and the combined extracts were dried over Na₂SO₄,filtered, and the filtrate was evaporated to dryness. Ethyl ether wasadded and the precipitate collected by filtration and dried under vacuumat room temperature overnight to yield 0.8 g of product as a whitepowder. The product had 79% substitution of the maleimidyl group on thePEG moiety. The ¹H nmr was consistent with that of N-maleimidyl methoxyPEG (nmr: dmso-d6: 3.51 ppm, PEG backbone; 7.03, CH═CH).

Example 2 Preparation of N-maleimidyl poly(ethylene glycol) Amine

tBoc PEG amine (MW 3400 Da, 2.0 g was dissolved in saturated aqueousNaHCO₃ (10 ml) and the mixture was cooled to 0° C.N-methoxycarbonylmaleimide (0.28 g) was added with vigorous stirring.After stirring for 10 minutes, water (20 ml) was added and the mixturewas stirred an additional 45 minutes. The pH was adjusted to 3.0 with0.5 N sulfuric acid and about 15 wt % NaCl was added. The reaction wasextracted with CH₂Cl₂ and the combined extracts were dried over Na₂SO₄,filtered, and the filtrate was evaporated to dryness. Ethyl ether (150ml) was added and the precipitate collected by filtration and driedunder vacuum at room temperature overnight to yield 1.5 g of the productas a white powder. The product had 74% substitution of the maleimidylgroup on the PEG moiety. The ¹H nmr was consistent with that ofN-maleimidyl methoxy PEG (nmr: dmso-d6: 3.51 ppm, PEG backbone; 7.03,HC═CH).

The above described crude N-maleimidyl PEG t-Boc amine was purified byion exchange chromatography. The crude product (1.45 g in 100 mldeionized water) was loaded onto DEAE Sepaharose, 100 ml) and elutedwith aqueous NaCl (15%, pH 3). The eluent was extracted with CH₂Cl₂(3×100 ml) and the extract was dried over Na₂SO₄, evaporated to drynessand precipitated with ethyl ether (100 ml). The precipitate wascollected by filtration and dried under vacuum at room temperature toyield 0.7 g of N-maleimidyl PEG t-Boc amine. The ¹H nmr was consistentwith that of N-maleimidyl PEG t-Boc amine (¹H nmr: dmso-d6: 1.37 ppm,t-butyl; 3.51, PEG backbone; 7.03, HC═CH).

N-maleimidyl PEG t-Boc amine (0.7 g) was dissolved in trifluoroaceticacid/CH₂Cl₂ (1:1, 20 ml) and stirred at room temperature for 1 h. Thesolution was evaporated to dryness under vacuum and the product wasprecipitated by the addition of ether (100 ml). The product was driedunder vacuum overnight to yield 0.58 g of N-maleimidyl PEG ammoniumtrifluoroacetate as a white powder. The product had 92% substitution ofthe N-maleimidyl group on PEG by GPC. The product was shown by ¹H nmr tohave about 100% substitution of the amine group on PEG and about 90% ofthe maleimidyl group on PEG. ¹H nmr (dmso d-6): 3.51 ppm, PEG backbone;7.03, HC═CH, 7.80, NH₃ ⁺.

Example 3 Preparation of N-maleimidyl poly(ethylene glycol) PropionicAcid and N-maleimidyl poly(ethylene glycol) N-succinimidyl Propionate

NH₂-PEG-O—CH₂CH₂CO₂H (MW 3400 Da, 2.0 g) was dissolved in a saturatedaqueous solution of NaHCO₃ (10 ml) and the mixture was cooled to 0° C.Powdered N-methoxycarbonylmaleimide (0.28 g, 3 eg.) was added withvigorous stirring. After stirring an addition 10 min., 20 ml of H₂O wasadded and the mixture was stirred for 45 min. at room temperature. ThepH was adjusted to 3 with 0.5 N H₂SO₄ and NaCl was added to aconcentration of about 15 wt %. The reaction mixture was extracted withCH₂Cl₂ (100 ml×3), dried over Na₂SO₄ and evaporated to dryness. Afterprecipitation with ether (150 ml), the product was collected byfiltration and dried under vacuum (yield: 1.8 g). Purity was 95% by GPC.¹H nmr (dmso-d6): 2.44 ppm, t, CH₂—CO₂—; 3.27, t, CH₂—N; 3.51, br s, PEGbackbone); 7.03, HC═CH.

N-maleimidyl-PEG-OCH₂CH₂CO₂H (1.0 g) was dissolved in CH₂Cl₂ (15 ml) andN-hydroxysuccinimide (0.042 g, 2 eq.) and DCC (0.074 g, 2 eq.) was addedand the solution was stirred overnight at room temperature. Theresulting mixture was concentrated under vacuum and the productprecipitated by addition of IPA:ether (1:1, 100 ml). The product waswashed with ether (30 ml) and dried under vacuum overnight (yield: 0.89g). ¹H nmr (dmso-d6): 2.81 ppm, s, —CH₂CH₂— on NS; 2.93, t, CH₂—CO₂;3.51, PEG backbone, br s; 7.03, HC═CH.

Example 4 Preparation of 4-Arm 10 KDa PEG Maleimide

4-Arm PEG amine (ave. MW=10,000 Da, 1 g) was dissolved in saturatedaqueous NaHCO₃ (5 ml) and the mixture was cooled to 0° C.N-methoxycarbonylmaleimide (0.2 g) was added with vigorous stirring.After stirring for 10 minutes, water (10 ml) was added and the mixturewas stirred an additional 45 minutes. The pH was adjusted to 3.0 with0.5 N sulfuric acid and about 15 wt % NaCl was added. The reaction wasextracted with CH₂Cl₂ and the combined extracts were dried over Na₂SO₄,filtered, and the filtrate was evaporated to dryness. Ethyl ether wasadded and the precipitate collected by filtration and dried under vacuumat room temperature overnight to yield 0.8 g of the product as a whitepowder. The product had 85% substitution of the maleimidyl group on thePEG moiety. The 41 nmr was consistent with that of N-maleimidyl 4-armPEG (nmr: dmso-d6: 3.51 ppm, PEG backbone; 7.03, CH═CH).

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of teachings presented in the foregoing descriptions and theassociated drawings. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosed andthat modifications and other embodiments are intended to be includedwithin the scope of the appended claims. Although specific terms areemployed herein, they are used in a generic and descriptive sense onlyand not for purposes of limitation.

That which is claimed is:
 1. A homobifunctional N-maleimidyl polymerhaving a formula:

wherein n is about 20 to about
 4000. 2. The homobifunctionalN-maleimidyl polymer of claim 1, wherein the polyethylene glycolbackbone has an average molecular weight of from about 800 daltons toabout 100,000 daltons.
 3. A method of forming a homobifunctionalN-maleimidyl polyethylene glycol polymer, the method comprising: (i)reacting a N-maleimidyl polyethylene glycol amine,

with maleic anhydride to form an open ring amide carboxylic acidintermediate,

wherein the polyethylene glycol backbone of the N-maleimidylpolyethylene glycol amine has an average molecular weight of from about800 daltons to about 100,000 daltons, and (ii) heating the open ringamide carboxylic acid intermediate in the presence of acetic anhydrideand an acetate salt to thereby form the homobifunctional N-maleimidylpolyethylene glycol polymer,


4. The method of claim 3, wherein the acetate salt in step (ii) issodium acetate.
 5. The method of claim 3, wherein the acetate salt instep (ii) is potassium acetate.
 6. The method of claim 3, wherein theheating in step (ii) is at a temperature from about 50° C. to about 140°C.
 7. The method of claim 3, wherein the heating in step (ii) is carriedout for about 0.2 hours to about 5 hours.
 8. The method of claim 3,further comprising purifying the homobifunctional N-maleimidylpolyethylene glycol polymer formed in step (ii).
 9. The method of claim8, wherein the purifying comprises precipitation.
 10. The method ofclaim 8, wherein the purifying comprises ion exchange chromatography.